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Phage display cycle. 1) fusion proteins for a viral coat protein + the gene to be evolved (typically an antibody fragment) are expressed in bacteriophage. 2) the library of phage are washed over an immobilised target. 3) the remaining high-affinity binders are used to infect bacteria. 4) the genes encoding the high-affinity binders are isolated.
A promising strategy for the construction of DNA-encoded libraries is represented by the use of multifunctional building blocks covalently conjugated to an oligonucleotide serving as a “core structure” for library synthesis. In a ‘pool-and-split’ fashion a set of multifunctional scaffolds undergo orthogonal reactions with series of ...
Construction of pre-made phage display libraries. Pre-made phage display library [10] is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages (viruses that infect bacteria) to connect proteins with the genetic information that encodes them.
The first step is to have phage display libraries prepared. This involves inserting foreign desired gene segments into a region of the bacteriophage genome, so that the peptide product will be displayed on the surface of the bacteriophage virion. The most often used are genes pIII or pVIII of bacteriophage M13. [5]
Bacterial display (or bacteria display or bacterial surface display) is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures (biopanning).
For example, the library size for phage and bacterial display is limited to 1-10 × 10^9 different members. The library size for yeast display is even smaller. Moreover, these cell-based display system only allow the screening and enrichment of peptides/proteins containing natural amino acids.
The resulting phage particles that are produced contain the single-stranded phagemids and are used to infect XL-1 Blue cells. [2] The double-stranded phagemids are subsequently collected from these XL-1 Blue cells, essentially reversing the process used to produce the original library phage.
Cell surface display systems can also be utilized to screen mutant polypeptide libraries. The library mutant genes are incorporated into expression vectors which are then transformed into appropriate host cells. These host cells are subjected to further high throughput screening methods to identify the cells with desired phenotypes. [5] [page ...